This invention pertains to the production and use of Pseudomonas-derived immunotoxins modified to increase their toxicity and potency in therapy. In particular, the immunotoxins of this invention include a disulfide-stabilized (ds) target-binding agent, such as the variable region of an antibody molecule, and a Pseudomonas exotoxin that does not require proteolytic activation for cytotoxic activity.
Immunotoxins were initially produced by chemically coupling antibodies to toxins (Vitetta et al. Cell, 41: 653-654 (1985); Pastan et al., Ann. Rev. Biochem. 61: 331-354 (1992)) to form chimeric molecules. In these molecules, the antibody portion mediated selective binding to target cells, while the toxin portion mediated translocation into the cytosol and subsequent cell killing. Several toxins have been used to make immunotoxins, including ricin A chain, blocked ricin, saporin, pokeweed antiviral protein, diphtheria toxin and Pseudomonas exotoxin A (PE) (Pastan et al., Science 254: 1173-1177 (1991); Vitetta et al., Semin. Cell Biol. 2: 47-58 (1991); Tazzari et al., Br. J. Hematol. 81: 203-211 (1992); Uckun et al., Blood, 79: 2201-2214 (1992)).
Several clinical trials with immunotoxins have shown activity against lymphomas and other cancers derived from the hematopoietic system (Vitetta et al., Cancer Res. 51: 4052-4058 (1991); Grossbard et al., J. Clin. Oncol. 11: 726-737 (1993)). However, these immunotoxins are heterogeneous and their large size limits penetration into solid tumors. Second generation immunotoxins are totally recombinant molecules made by fusing the smallest functional module of an antibody, the Fv fragment, to a truncated toxin which lacks the cell-binding domain (Brinkmann et al., Proc. Natl. Acad. Sci. USA 88: 8616-8620 (1991); Kreitman et al., Blood, 80: 2344-2352 (1992)). The small size of single-chain Fv-immunotoxins makes them much more useful than chemical conjugates of whole antibodies for certain therapeutic applications because their small size increases tumor penetration and efficacy (Fukimori et al., Cancer Res. 49: 5656-5663 (1989); Jain, Cancer Res., 50: 814-819 (1990); Sung et al., Cancer Res. 50: 7382-7392 (1990)).
Several types of recombinant Fv-immunotoxins containing PE have been made and tested in vitro as well as in animal models (Brinkmann et al., Proc. Natl. Acad. Sci. USA 88: 8616-8620 (1991); Kreitman et al., Blood, 80: 2344-2352 (1992); Batra et al., Proc. Natl. Acad. Sci. USA 89: 5867-5871 (1992); Reiter et al., Cancer Res. 54: 2714-2718 (1994); Brinkmann et al., Proc. Natl. Acad. Sci. USA 90: 547-551 (1993)). Initially, the Fv regions of the immunotoxins were arranged in a single-chain form (scFv-immunotoxin) with the V.sub.H and V.sub.L domains connected by a linking peptide. More recently, disulfide-stabilized forms of Fv-immunotoxins (dsFv-immunotoxins) have been generated in which the V.sub.H and V.sub.L domains are connected by a disulfide bond engineered into the framework region (see, e.g. copending application U.S. Ser. No. 08/077,252 filed on Jun. 14, 1993; Brinkmann et al., Proc. Natl. Acad. Sci. USA 90: 7538-7542 (1993); Reiter et al., Protein Eng., 7: 697-704 (1994)). Disulfide-stabilized Fv immunotoxins are much more stable than single-chain immunotoxins and can show improved binding to antigen (Reiter et al., J. Biol. Chem. 269: 18327-18331 (1994); Reiter et al., Protein Eng. 7: 697-704 (1994)). In addition, dsfv-immunotoxins are slightly smaller in size than scfv-immunotoxins, and may exhibit better tumor penetration.
Recombinant immunotoxins containing PE must be proteolytically activated within the cell by cleavage in domain II between amino acids 279 and 280 (Ogata et al. J. Biol. Chem., 267: 25369-25401 (1992)). To eliminate the need for intracellular proteolytic activation and thereby increase cytotoxic activity, the toxin moiety of recombinant toxins has been modified. This was initially done with recombinant toxins containing TGF.alpha. by producing a truncated toxin (PE280-613) with TGF.alpha. inserted near the end of domain III at position 607 (Theuer et al., J. Urol., 149: 1626-1632 (1993); Theuer et al., Cancer Res., 53: 340-347 (1993)). Because the toxin begins at position 280, it does not need proteolytic activation within the cell (Ogata et al. J. Biol. Chem., 267: 25369-25401 (1992); Theuer et al. J. Biol. Chem., 267: 16872-16877 (1992)). In addition, these molecules had two other mutations. One was a deletion of unnecessary residues in domain Ib (365-380). The other was to change the carboxyl terminus from REDLK (SEQ ID NO:8) to KDEL (SEQ ID NO:9) to increase cytotoxic activity (Seetharam et al. J. Biol. Chem., 266: 17376-17381 (1991)). This molecule termed PE35/TGF.alpha.KDEL was 10-700 fold more active than TGF.alpha.-PE40 on several human bladder cancer cell lines (Theuer et al., J. Urol., 149: 1626-1632 (1993)). However, even more specific and reactive immunotoxins are desired.